The present invention relates to a method for analyzing the primary structure of a protein or peptide. Specifically, the present invention relates to a method for sequencing from N-terminal of the protein or peptide, whose .alpha.-amino group in the amino terminal (N-terminal) amino acid residue is modified.
It has been clarified that the N-termini of many proteins are modified. It is reported that more than half of the proteins receive some kinds of modifications on the N-termini. Generally, Edman degradation is used for sequencing of protein from the N-terminal amino acid. However, the proteins having modified N-termini cannot be sequenced by Edman degradation.
For example, there are modified proteins (N-acetyl proteins) which have an acetylated .alpha.-amino group in the N-terminal amino acid residue. Among proteins which are modified at the N-termini, 80% or more are said to be acetylated. Furthermore, among the N-terminal acetylated proteins, approximately 30% are those to be acetylated at the .alpha.-amino group in the N-terminal serine (N-acetylseryl protein), and approximately 10% are those to be acetylated at the .alpha.-amino group in the N-terminal threonine (N-acetylthreonyl protein).
Conventionally, to analyze the sequence of amino acids from the N-terminal amino acid of these N-acetyl proteins, the following methods have been reported.
Conventional Method 1!
(1) Digest an N-acetyl protein with a protease to thereby prepare a mixture of plural peptide fragments. PA0 (2) Allow .alpha.-amino groups in newly generated peptide fragments to react with phenylisothiocyanate to thereby form thiocarbamyl compounds. PA0 (3) Oxidize the thiocarbamyl compounds with a performic acid to form carbamyl compounds so that they are inert during the process of Edman degradation. PA0 (4) Use an acetylamino acid releasing enzyme to free the acetylated N-terminal amino acid in the N-terminal peptide of the original sample protein. PA0 (5) Thereafter, analyze the sequence of the amino acids by Edman degradation. PA0 (1) Digest an N-acetyl protein with a protease to thereby prepare a mixture of plural peptide fragments. PA0 (2) Purify peptide fragments using gel filtration. PA0 (3) From the mixture of peptide fragments, fractionate the N-terminal peptide of the original protein sample using HPLC. PA0 (4) Use an acetylamino acid releasing enzyme to free the acetylated N-terminal amino acid from the N terminal peptide of the original sample protein. PA0 (5) Then sequentially analyze the sequence of amino acids by Edman degradation. (Zoku Seikagaku Jikkenn Kouza 2, Tanpakushitu No Kagaku (Vol.1), Edited by Nihon Seikagakukai, Tokyo Kagaku Dojin, p227)
Here, the N-terminal amino acid of an N-terminal peptide of an original sample protein had been already acetylated and thus do not form a thiocarbamyl compound.
In this method, only the N-terminal peptide of the original protein sample is subjected to Edman degradation. (Tsunazawa et al., J. Protein Chem. (1992) vol.11, p382)
Conventional Method 2!
Conventional Method 3!
Perform operations 1 to 3 in the Convention Method 2.
Analize thus obtained N-terminal peptide of the original sample protein by way of mass spectrometry to analyze the sequence of the amino acid (Zoku Seikagaku Jikkenn Kouza 2, Tanpakushitu No Kagaku (Vol. 1), Edited by Nihon Seikagakukai, Tokyo Kagaku Dojin, p228).
The conventional methods have the following disadvantages: (1) since they require strong reaction conditions, derivatization of samples occur, (2) since they require troublesome operation steps including the use of special devices, thereby being low in yield, and (3) since they use enzymes which have several substrate specificity and activity, an accurate analysis becomes difficult to obtain.